1. Field of the Invention
The present invention relates to methods for producing substantially homogenous cell populations of functionally differentiated cells, preferably antigen-presenting dendritic cells or polymorphonuclear neutrophils, from hematopoietic cells.
2. Technical Background
Differentiation of hematopoietic cells involves the highly ordered and controlled proliferation of immature progenitor cells and their commitment and differentiation into fully mature cells of various lineages. While a number of retroviral oncogenes efficiently bypass such normal control mechanisms and cause leukemia, they also provide invaluable tools to study mechanisms of normal hematopoietic cell differentiation on the molecular level. In the avian system, oncogene transformed, non established cell strains can be obtained in vitro under conditions where they retain their capacity to undergo apparently normal terminal differentiation (reviewed in Beug and Graf, 1989).
v-rel, the oncogenic version of c-rel transduced by the avian retrovirus REV-T/REV-A, belongs to the NF-.kappa.B/rel/dorsal transcription factor family (reviewed by Gilmore, 1991). Members of this still growing protein family are versatile regulators involved in growth control, differentiation and pattern formation. v-rel encodes a 59 kd protein which forms multiple complexes with several other cellular proteins (Morrison et al., 1989; 1992; Gilmore, 1991 and references therein) and transforms avian hematopoietic cells both in vivo and in vitro (reviewed in Bose, 1992).
Several groups have demonstrated that v-rel acts as a transcriptional repressor of rel- and/or NF-kB-responsive genes in transient transfection assays (Ballard et al., 1990; lnoue et al., 1991; Richardson and Gilmore, 1991; McDonnell et al., 1992; Ballard et al., 1992). Studies with a conditional hormone-inducible v-rel/estrogen receptor fusion protein (v-relER), however, provided first clues as of v-rel acting as a transcriptional activator of rel- and/or NF-.kappa.B-responsive genes in transformed bone marrow cells (Boehmelt et at., 1992). Such a hormone-inducible v-relER caused estrogen-dependent but otherwise unaltered v-rel-specific transformation of chicken bone marrow cells in vitro.
Initial evidence suggested that v-rel contained within the REV-T/REV-A virus complex induced a disease of lymphomatous origin (Sevoian et al., 1964). This is in line with the observation that oncogenic activation of other members of the NF-.kappa.B/rel/dorsal family (e.g., Lyt-10/NF-.kappa.B2) has been implicated in lymphoid tumor formation in humans (Neri et al., 1991; Lu et al., 1991; Fracchiolla et al., 1993). The in vitro target cell for v-rel transformation was also classified as lymphoid, more specifically, as early preB- or preB/preT-lymphoid progenitor (Beug et al., 1981; Lewis et al., 1981). Finally, REV-T transformation carried out with helper viruses other than REV-A appeared to be of B-lymphoid origin (Barth and Humphries, 1988; Benatar et al., 1991, 1992; Bose, 1992).
Histological studies, however, suggested that the REV-T/REV-A-induced disease was reticuloendotheliosis (Theilen et al., 1966; Olson, 1967) rather than lymphomatosis, since cells of the reticuloendothelial system (RES) were affected. RES is a collective term for a system of scattered phagocytic cells associated with endothelia of blood vessels and with sinusoids of spleen, liver and lymphoid organs. Cells of the RES are also dispersed in the connective tissue surrounding endothelia and were described to exhibit a high degree of flexibility and mobility.
However, there are few studies in line with these histological characterizations. Barth et al. (1990) showed that the tissue derived from liver or spleen tumors of REV-T/REV-A infected chickens yields transformed cells exhibiting either T-lymphoid or myeloid determinants as demonstrated by surface antigen expression. Using a replication competent virus containing v-rel, Morrison et al. (1991) demonstrated that v-rel transformed chicken bone marrow cells coexpress surface antigens specific to both lymphoid and myeloid cells. Thus, the true target cell for v-rel transformation remained obscure.
Coexpression of lineage-specific cell surface markers is often observed on human leukemic cells, probably due to aberrant gene expression associated with the leukemic phenotype (McCulloch, 1983; Greaves et at., 1986 and references therein). By analogy, coexpression of both myeloid- and lymphoid-specific surface antigens on v-rel transformed cells could be the result of aberrant gene expression induced by the active v-rel oncogene. Alternatively, as suggested before, v-rel transformed cells might represent early, potentially bi- or pluripotent hematopoietic progenitors with the capacity to differentiate into two (or more) mature cell types (Morrison et al., 1991; Boehmelt et al., 1992).
Dendritic cells are found at various locations within an organism and have been classified as e.g., dendritic/Langerhans cells in the skin, "veiled" cells, interdigitating cells, or follicular dendritic cells, depending on their presumptive function and location (Steinman et al., 1991). They are considered to capture antigens and migrate to lymphoid organs where they present the processed antigens to lymphoid cells. While it is well established that they represent professional antigen-presenting cells, it is still a matter of debate how the different types of dendritic cells relate to each other.
Dendritic cells have been obtained from peripheral blood, bone marrow, spleen (Inaba et al., 1992, 1993) and by in vitro differentiation from CD34+ human peripheral blood stem cells (Caux et al., 1992). A detailed analysis of their functional and biochemical properties has remained difficult, mainly because pure and homogenous cell populations are not yet available and because of the limited cell numbers obtained. Very recently, a v-myc transformed immortalized mouse cell line with features of dendritic cells (or of a late dendritic cell progenitor) was described (Paglia et al., 1993) which should facilitate the analysis of mouse dendritic cells in vitro. Additionally, specific culture conditions were developed for propagation of human dendritic cells, which, however, rapidly slowed down in growth after 3 weeks in culture (Romani et al., 1994; Sallusto a Lanzavecchia, 1994).
To achieve a better understanding of molecular mechanisms of disease and for the search of new drugs, there is a need for in vitro models of vertebrate cell systems, especially culture systems, containing well-defined, homogenous populations of differentiated cells which exhibit all or all essential parts of their normal physiological function. Transformation of immature hematopoietic cells with a regulatable oncogene product, a conditionally active v-rel (e.g., v-relER; Boehmelt et al., 1992; Capobianco and Gilmore, 1993) has opened the possibility of reversibly inducing growth of bone marrow cells and concomitantly blocking their differentiation. However, while such cells can be expanded in vitro under specific culture conditions, methods for inducing cellular differentiation have not appeared in the literature.